Figure 3
Activity profile of the double mutant KLK7 (dmKLK7). (a) Surface exposed residues corresponding to the catalytic triad of dmKLK7 (His57, Asn102 and Ala195) are shown on the 3D-structure available in PDB (accession: 2QXG.pdb in standard serine protease orientation) using SPDBV v4.10. Alongside, are the peptides identified in the MS-analysis (underlined) aligned with the KLK7 full length protein (UniProtKB; P49862-1). S195A and D102N mutations are as highlighted in red bold letters. (b) dmKLK7 cleavage site preferences in the tryptic-PICS library (1:50; enzyme/library) in the form of a 2D-heat map showing scattered cleavage specificities from P6-P6′ with no chymotryptic-like cleavage site specificities. (c) Casein-zymogram comparing active KLK7, mKLK7 and dmKLK7; (d) MeO-Suc-Arg-Pro-Tyr-MCA peptide substrate assay (n = 6, mean ± SD) with mKLK7 and dmKLK7; and (e) silver-stained SDS-PAGE with the protein substrate, FN, comparing active KLK (1/10 to 1/1000) with dmKLK7 .
